1. Field of the Invention
The present invention provides an avian recombinant herpesvirus comprising cDNA of VP2 (VP2 cDNA) of the IBDV Delaware Variant E strain, a member of IBDV variant strain subtype.
2. Related Art
Infectious Bursal Disease, often referred to as Gumboro disease, is caused by the highly transmissive Infectious Bursal Disease Virus (IBDV) and inflicts a great economic loss on the poultry industry. There are two serotypes of IBDV: serotype 1 and 2. Serotype 1 is pathogenic for chickens. Many IBDV strains are classified as serotype 1. Based on pathogenicity and antigenicity, serotype 1 strains are further divided into four subtypes: classical virulent, attenuated, variant, and very virulent strains (Y. C. Cao et al., 1998, Avian Diseases, 42: 340–351). For instance, STC, 52/70, 002–73, etc., are classified as classical virulent strains. Bursine, Bursa-Vac-3. Bursa-Vac-M, CU-1, PBG98, etc., are classified as attenuated strains. Delaware E, A, GLS, GZ902, Ark, AL-2, etc., are classified as variant strains, and OKYM, UK661, F9502, HK46, etc., are classified as very virulent strains.
The IBDV genome consists of two double-stranded RNA segments: Segment A and B. Segment A encodes a 115 kd precursor polyprotein, which is processed auto-catalytically by VP4 to give VP2, VP3 and VP4. Segment A also encodes VP5 that is translated in a different reading-frame from VP2 to VP4. Reportedly, VP5 is related to the virulence of the virus but its detailed function remains to be elucidated. Earlier studies using anti-IBDV monoclonal antibodies indicate that VP3 has one serotype-specific epitope and another non-overlapping epitope, but VP3 does not contain a major virus-neutralizing epitope. On the contrary, VP2 has independent epitopes that elicit IBDV neutralizing antibodies (U.S. Pat. No. 5,350,575, U.S. Pat. No. 5,849,575). The amino acid sequence of VP2 differs from strain to strain (H. G. Heine et al., 1991, J.Gen.Virol. 72: 1835–1843, T. V. Dormitorio et al., 1997, Avian Diseases 41:36–44, and Y. C. Cao, et al., 1998, Avian Diseases 42: 340–351), and antigenic differences among IBDV subtypes are likely due to these sequence variances.
In the United States, Infectious Bursal Disease has been controlled by passive immunity passed from the hen to the chick. In short, high antibody levels are elicited using killed IBDV vaccines so that chicks acquire high maternal antibody levels. These high maternal antibody levels protect the chick through the first few weeks of life. Problems associated with this strategy are that all chicks do not acquire the same level of maternal antibody. On a flock basis it is hard to predict when maternal antibodies wane and as a result some chicks are unprotected. In the United States in the 1980s, variant IBD viruses breaking through passive immunity elicited with classic vaccine strains caused disease in the United States (H. G. Heine et al., 1991, J. Gen. Virol. 72:1835–1843). The most prominent variant virus at this time was Delaware Variant E. Variant IBDV viruses were and are still being added to killed vaccines for protection against variant strains. In Europe, many cases of Infectious Bursal Disease were reported among chickens that had high titers of IBDV maternal antibody. These chickens were killed by the natural infection of a very vilulent strain even though hens were administered IBD vaccines (M. D. Brown et al., 1994 J. Gen. Virol. 75:675–680). These incidents indicate that antigenic differences between vaccine and prevalent disease-causing strains should be seriously considered. Development of a vaccine that protects chickens from a variety of different subtypes of IBDV is desirable for the poultry industry.
Construction of a recombinant avian herpesvirus harboring a protective antigen gene from other avian pathogens as well as its use as a poultry vaccine is suggested in U.S. Pat. No. 5,834,305, U.S. Pat. No. 5,853,733, U.S. Pat. No. 5,928,648, U.S. Pat. No. 5,961,982, WO 87/04463 and WO 99/18215 etc. VP2 is a protective antigen of IBDV and Segment A of IBDV includes VP2 gene. The recombinant avian herpesvirus comprised of the VP2 gene or Segment A and its use as an IBD vaccine is reported in U.S. Pat. No. 5,733,554, WO 89/01040, WO 93/95665, WO 96/05291 or WO99/18215. R. Darteil et al., and K. Tsukamoto et al. also reported similar recombinant avian herpesvirus-vectored IBD vaccines (R. Darteil et al., 1995, Virology, 211:490–490. K. Tsukamoto et al., 1999, Virology, 257:352–362). In WO 89/01040, under the control of the pseudorabies virus gpX promoter, cDNA of Segment A including VP2, VP3 and VP4 genes was inserted into the BamHI #16 fragment in the UL43 gene of herpesvirus of turkeys (HVT) to generate a recombinant herpesvirus, S-HVT-003. Segment A was derived from the IBDV S4047 strain but its subtype is not divulged in the specification. In addition, R. Darteil et al. (R. Darteil et al., 1995, Virology, 211:481–490. U.S. Pat. No. 5,733,554) reported a few recombinant HVTs harboring VP2 gene from the IBDV 52/70 strain, a member of the classical virulent strain subtype. For instance, vHVT1 comprises the said VP2 gene in the RR2 (UL40) region, which is driven by the RR2 intrinsic promoter. vHVT2 comprises the VP2 gene under the control of the exogenous CMV-1E promoter in the gl (US7) region. vHVT4 comprises the VP2 gene driven by the same promoter in the UL43 region. Although the UL40 and US7 regions seemed not to be essential for in vitro virus growth, vHVT1 and vHVT2 did not grow well in vivo. On the contrary, vHVT4 conferred good protection in SPF chickens against challenge with the IBDV 52/70 strain. However, in this experiment, the challenge conditions seem to have been mild since the group of positive control chickens, vaccinated with an inactivated IBDV vaccine, was also completely protected. Inactivated vaccines do not induce protective immunity against very virulent strains or European types of virulent strains.
In addition, WO 99/18215 describes a recombinant HVT, HF003, which has the VP2 gene inserted into the inter-ORF region between UL45 and UL46. The said VP2 gene was from IBDV OKYM, a member of the very virulent strain subtype, which was isolated in Japan. However, HF003 was proven to confer protection only against the IBDV OKYM strain.
In consequence, several avian recombinant herpesviruses comprising IBDV genes have been reported so far, but none of these induced in chickens protective immunity against a variety of different subtypes of IBDV. In other words, no knowledge is available as to which VP2 gene is suitable for the construction of the recombinant avian herpesvirus that will give protection against the broad range of IBDV subtypes.